Hemophilias A and B are two diseases for which a cure was predicted some years ago using gene-based therapeutic approaches. This enthusiasm was due in part to the relative simple clinical endpoints required for disease correction, and the availability of a large animal model which recapitulated the human disorder. Even though the preclinical strategies our laboratory developed were used to obtain long-term amelioration of the bleeding diathesis in canine hemophilia B, the direct translation of this strategy in humans was not successful. It is now more reasonable to speculate that small incremental improvements in a subset of patients will likely be realized in the more immediate future. Therefore there is still a great need to continue translational-based research towards finding an approach that can result in a cure in all patients. We believe that the two most promising vector systems for treating the hemophilias are recombinant Adeno-associated viral vectors (rAAV) and non-viral DNA plasmid-based vectors. We propose to continue our studies with these two vector systems and work towards their pre- clinical development. We plan to pursue a new strategy for obtaining AAV-mediated integration of the factor IX mini-gene into the ribosomal DNA locus, a benign but yet robust site for AAV- mediated transgene expression. We will use this approach for preclinical testing in both mice and dogs. We propose new studies to produce high-grade pure minicircle DNA vectors, an episomal gene transfer system we have developed and used to achieve long-term transgene expression in vivo. We will further explore the mechanism(s) involved in determining how minicircle-DNA is more robust at maintaining transgene expression in vivo compared to routine plasmids. To do this, we will study the nucleosome structure of different DNA vectors delivered into mouse liver. Finally, we plan to screen and evaluate nanoparticle approaches for clinically relevant gene transfer into the liver in preclinical animal trials. We believe these approaches will substantially enhance our ability to use gene transfer to treat the hemophilias. As in our previous studies with AAV vectors, we believe these studies will be useful for developing new clinical trials for hemophilia.

Public Health Relevance

Hemophilias A and B have been considered a good target for gene transfer-based treatment because of the relative simplicity required for achieving a cure. Several vectors offer great potential for some gene therapy applications but, after a number of years in early clinical trials and despite success in animal models, there is no established successful gene therapy treatment for hemophilia. We plan to continue our studies on non-viral gene transfer vectors by improving their purity, establishing how they maintain their activity in vivo long-term, and developing a clinically relevant DNA delivery strategy. Although rAAV vectors have been used by our group in clinical trials, sustained transgene expression was not obtained in humans, unlike in animal models. We plan to pursue a strategy for site-specific integration of the recombinant AAV-based transgene expression cassette into a benign yet robust region of the chromosome, making a single administration of this vector last for life. Our studies will advance gene therapeutic approaches towards achieving a cure for the hemophilias.